Godfrey E. Etokebe

Hip osteoarthritis susceptibility is associated with IL1B -511(G>A) and IL1 RN (VNTR) genotypic polymorphisms in Croatian Caucasian population

Among the predisposing factors to osteoarthritis (OA), a frequent destructive joint disease, is the complex genetic heritage including the interleukin‐1 family members like the IL1β (IL1B) and the IL1 receptor antagonist (IL1RN) genes. The aim of this study was to investigate allelic and genotypic frequencies of the IL1B gene single nucleotide polymorphism (SNP) at −511(G>A) and the variable number tandem repeat (VNTR) in the IL1RN gene in a Croatian Caucasian population of hip OA (HOA) cases and healthy controls. A total of 259 HOA patients with total hip replacement (THR) and 518 healthy blood donors as controls were genotyped for IL1B gene SNP −511(G>A) and the VNTR in the IL1RN gene associated with HOA. The genotype G/A (1/2) at IL1B was significantly associated with the protection of the HOA (p < 0.036, OR = 0.72, 95% CI = 0.52–0.99). The genotype G/G (1/1) had only a trend towards the susceptibility (p = 0.053, OR = 1.35, 95% CI = 0.98–1.86) to disease. None of the haplotypes IL1B −511(G>A) and IL1RN (VNTR) were found associated with the HOA. The haplotype 1–2 at these loci had only a trend to susceptibility (p = 0.065). Haplotype 1–3 had a significant male bias in diseased. Furthermore, genotype comprising 2–1/2–2 haplotypes was found significantly associated with predisposition to HOA (p = 0.027, OR = 2.23, 95% CI = 1.03–4.88), whereas genotype 1–1/2–2 with protection to disease (p = 0.028, OR = 0.65, 95% CI = 0.43–0.97). Our findings suggest that HOA in Croatian population might have a different genetic risk regarding the IL1 locus than has been reported for other Caucasian populations previously.

Interferon-γ Gene (T874A and G2109A) Polymorphisms Are Associated With Microscopy-positive Tuberculosis

Genetic susceptibility to tuberculosis includes several unknown yet different loci each contributing to a small extent. Intronic polymorphisms within the interferon‐γ (IFN‐γ) gene IFNG T+874A and IFNG G+2109A correlate with the IFN‐γ production in vitro, and the frequency of potential high IFN‐γ producers was previously reported by others to be lower in patients than in controls from Sicily. The aim of this study was to determine whether there is an association between polymorphisms in the IFN‐γ gene and predisposition to tuberculosis. We analysed two IFNG SNPs (T+874A and G+2109A) in patients (n = 253) hospitalized in Rijeka (Croatia) and controls (n = 519) from the same area. One‐fifth of the controls were healthy contacts of the diseased, and the rest were blood donors. IFNG alleles, their predicted haplotypes or genotypes were not associated with disease susceptibility. Thus, we could not reproduce results from Sicilian case‐control study. However, T/T+874 (possible high IFN‐γ producer) and +874A/A (putative low producer) genotypes were associated with microscopically positive–negative forms of disease. Haplotypes (T+874A and G+2109A) based on a prediction by software phase and subsequent genotype analysis corroborated these findings. Patients had significantly higher frequency of genotypes without T at +874 (AA/AA; AA/AG and AG/AG) in microscopy‐ or bacterial culture‐positive groups compared with their negative counterparts. These data suggest an association with disease severity rather than susceptibility to tuberculosis in Croatian Caucasian population.

Interferon-γ Receptor-1 Gene Promoter Polymorphisms (G-611A; T-56C) and Susceptibility to Tuberculosis

We analysed frequencies of two single‐nucleotide polymorphisms (SNP) in the interferon‐γ (IFN‐γ) receptor‐1 (IFNGR1) gene promoter (G‐611A, T‐56C) in tuberculosis patients (n = 244) and compared them with controls (n = 521). These frequencies were not significantly different, whether analysed independently or as haplotypes. Because these SNP affect transcription, the results suggest that the expression of the IFNGR1 gene does not confer susceptibility to disease in patients from Croatia. Further analysis revealed a significant association between the protective (CA)n polymorphism (22 repeats, 192 FA1), located in the fifth intron of the IFNGR1 gene (+16682), and GT promoter haplotype (−611; −56) that showed the strongest expression capacity. In addition to this cis relationship, the (CA)22 allele was correlated in trans with an IFN‐γ SNP (IFNG G + 2109A), which might affect the transcription of the IFNG gene. These results suggest that a particular combination of IFNG and IFNGR1 SNP might offer a better protection against tuberculosis in this population.

Toll-like receptor 2 (P631H) mutant impairs membrane internalization and is a dominant negative allele.

We have sequenced 416 Toll‐like receptor‐2 (TLR2) alleles in 208 subjects in a tuberculosis case–control study in Croatian Caucasian population. We found ten single nucleotide polymorphisms (SNP) among which three were novel (S97S, T138I and L266F). The genotype containing TLR2‐P631H SNP was significantly overrepresented in patients with tuberculosis when compared to contact controls, suggesting a small yet increased risk to disease. The causative agent of tuberculosis is Mycobacterium tuberculosis, which can bind to TLR2 with its lipoprotein coat. The TLR2‐P631H mutant has a dominant negative effect on the wild type TLR2 signalling in transfected HEK293 kidney cells using the NF‐κB‐driven luciferase as a reporter gene with ligands like M. avium extracts, Pam3CysSK4 or FSL‐1 that bind TLR2/TLR1 or TLR2/TLR6 heterodimers, respectively. Studies on internalization from the Regular Madine Darby Canine Kidney cell surface into the early endosomal compartments showed a lower rate of the mutant compared to the wild type. Our data, in combination with a report by others show that the TLR2‐P631H allele could be associated with protection to meningococcal meningitis, suggest that by dominantly inhibiting the response of cells important in the immune response this mutant might confer either protection or susceptibility to meningitis or tuberculosis, respectively.

Single-Nucleotide Polymorphisms in Genes Encoding Toll-Like Receptor -2, -3, -4, and -9 in Case–Control Study with Breast Cancer

BACKGROUND Genetic susceptibility to cancer is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting cancer. AIM OF THE STUDY Single-nucleotide polymorphisms (SNPs) of Toll-like receptor (TLR) 2, TLR3, TLR4, and TLR9 genes, which are important for innate immunity, were analyzed for the association with breast cancer. METHODS The SNPs comprised TLR2 (c.597T>C), TLR2 (c.1350T>C), TLR3 (c.1377C>T), TLR4 (c.896A>G), and TLR9 (c.1635A>G). The allelic and genotypic frequencies of these TLR SNPs were compared between patients (n = 130) and controls (n = 101) in a case-control study from Croatia. RESULTS TLR SNPs were not significantly different. From the population genetics viewpoint, we found that a hypomorphic variant of TLR4 (p.Asp299Gly) allele has no specific allelic frequency (8.4%) in the Croatian population (n = 496) compared to other Caucasians (6.5-10%). CONCLUSION These results suggest that polymorphisms in tested TLR genes are not likely to be associated with increased risk for developing breast cancer.

Genetic Polymorphisms in the Toll-like Receptor 10, Interleukin (IL)17A and IL17F Genes Differently Affect the Risk for Tuberculosis in Croatian Population.

Proinflammatory conditions leading to activation of macrophages via interferon‐γ bear an important role in host defence against intracellular bacteria such as Mycobacterium tuberculosis (Mt). Interleukin‐17 plays a similar role, as it appears to be also an activator of macrophages. Recently, the TLR‐10 was identified as an anti‐inflammatory factor that exerts its action via association with the TLR‐2 chain at the cell surface of macrophages, the latter being an Mt‐binding protein. We have previously found that gene polymorphisms that either inactivate the TLR2 gene product or have a dominant‐negative role are associated with tuberculosis (TB) in Croatian population. We have now extended our survey and found that single nucleotide polymorphism (SNP) in TLR10 (rs11096957) is associated with risk for TB. Homozygotes carrying the A allele are associated with predisposition to disease as analysed by the dominant model of inheritance. In contrast, SNPs in the proinflammatory IL17A and IL17F genes (rs2275913 and rs763780, respectively), found previously to correlate with the disease occurrence in Chinese population, were not significantly associated with tuberculosis in the Croatian population.

Associations of the Interleukin-1 Gene Locus Polymorphisms with Risk to Hip and Knee Osteoarthritis : Gender and Subpopulation Differences.

Genetic predisposition to the complex hereditary disease like osteoarthritis (OA) of the large joints (hip and knee) includes the interleukin‐1 gene (IL‐1) cluster on chromosome 2. Using a case–control study with 500 OA patients (240 knee and 260 hip OA patients, all with joint replacement), we analysed frequencies of IL‐1 gene cluster polymorphisms in Croatian Caucasian population. The control samples came from 531 healthy individuals including blood donors. We genotyped two single nucleotide polymorphisms in the IL‐1 gene locus at IL‐1A (−889, C>T, rs1800587) and IL‐1B (+3594, C>T, rs1143634) and compared their frequencies between patients and controls. We predicted haplotypes by combining current data with our previous results on gene polymorphisms (IL‐1B, rs16944 and the IL‐1 receptor antagonist gene [IL‐1RN] variable number tandem repeat [VNTR]) for the same population. Haplotype analyses revealed gender disparities and showed that women carriers of the 1‐2‐1‐1 haplotype [IL‐1A(rs1800587) – IL‐1B(rs1143634) – IL‐1B(rs16944) – IL‐1RN(VNTR)] had sixfold lower risk to develop knee OA. However, carriers of the 1‐1‐1‐2 haplotype of both sexes had over twofold higher predisposition to hip OA. Our results differ from some earlier studies in Caucasian subpopulations, which may be due to the fact that this is the first study to separate genders in assessing the IL‐1‐locus genetic risk of OA. The results suggest that inflammatory mediators like IL‐1 might be implicated in the pathogenesis of primary OA in large joints and that as yet unidentified gender‐specific factors exist in a Croatian Caucasian population.

Association of variable number of tandem repeats in the coding region of the FAM46A gene, FAM46A rs11040 SNP and BAG6 rs3117582 SNP with susceptibility to tuberculosis.

We analyzed for association between the Family with sequence similarity 46, member A (FAM46A) gene (located on chromosome 6q14.1), BCL2-Associated Athanogene 6 (BAG6) gene (located on chromosome 6p21.3) and tuberculosis in Croatian Caucasian. We genotyped the FAM46A rs11040 SNP, FAM46A VNTR and BAG6 rs3117582 polymorphisms in a case-control study with 257 tuberculosis patients and 493 healthy individuals in a Croatian Caucasian population. We found that genotype FAM46A 3/3 (three VNTR repeats homozygote) was associated with susceptibility to tuberculosis (p<0.0015, Pcorr.<0.029, Odds ratio = 2.42, 95% Confidence Interval = 1.34–4.3). This association suggests that the protein domain encoded by the VNTR might be important for the function of the FAM46A protein, which, in turn, could be relevant in developing tuberculosis. In addition, we found that FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotype 132 (G-3-C) is associated with susceptibility to tuberculosis (p<0.012, pcorr.<0.024, Odds ratio 3.45, 95% Confidence Interval = 1.26–9.74). This may suggests that the interaction between the FAM46A and BAG6 proteins may be involved in tuberculosis etiology. We found also that infection of human macrophages with heat-killed M. tuberculosis (H37Rv) led to over-expression of FAM46A (VNTR 3/4) transcript. This is the first study to show associations between the FAM46A gene VNTR polymorphisms, FAM46A rs11040 SNP:FAM46A VNTR:BAG6 haplotypes and any disease.

Susceptibility to large‐joint osteoarthritis (hip and knee) is associated with BAG6 rs3117582 SNP and the VNTR polymorphism in the second exon of the FAM46A gene on chromosome 6

Family with sequence similarity 46, member A (FAM46A) gene VNTR and BCL2‐Associated Athanogene 6 (BAG6) gene rs3117582 polymorphisms were genotyped in a case–control study with 474 large‐joint (hip and knee) osteoarthritis (OA) patients and 568 controls in Croatian population by candidate‐gene approach for association with OA. We found that BAG6 rs3117582 SNP genotypes were associated with protection (major allele homozygote) and susceptibility (major–minor allele heterozygote) to OA. BAG6 rs3117582 major allele (A) was associated with reduced risk to OA while the minor allele (C) was associated with increased risk to OA. We identified 6 alleles harboring 2 to 7 repeats making 20 genotypes for FAM46A. A rare FAM46A VNTR genotype comprising VNTR alleles with four and seven repeats (c/f) was associated with increased OA risk in both genders. The genotype with four and six repeats (c/e) was also associated with increased risk to OA in males. A polymorphic FAM46A allele with six repeats (e) was associated with reduced risk to OA in females. Our results suggest association between the FAM46A gene, BAG6 gene and OA in Croatian population, respectively. This is the first study to show associations between these genetic loci and OA.

Association of the FAM46A gene VNTRs and BAG6 rs3117582 SNP with non small cell lung cancer (NSCLC) in Croatian and Norwegian populations

We analyzed for associations between a variable number of tandem repeat (VNTR) polymorphism in the Family with sequence similarity 46, member A (FAM46A) gene and a single nucleotide polymorphism (rs3117582) in the BCL2-Associated Athanogene 6 (BAG6) with non small cell lung cancer in Croatian and Norwegian subjects. A total of 503 (262 Croatian and 241Norwegian) non small cell lung cancer patients and 897 controls (568 Croatian and 329 Norwegian) were analyzed. We found that the frequency of allele b (three VNTR repeats) of FAM46A gene was significantly increased in the patients compared to the healthy controls in the Croatian and the combined Croatian and Norwegian subjects. Genotype frequencies of cd (four and five VNTR repeats) and cc (four VNTR repeats homozygote) of the FAM46A gene were significantly decreased in the patients compared to the healthy controls in the Croatian and Norwegian subjects, respectively. Logistic regression analyses revealed FAM46A genotype cc to be an independent predictive factor for non small cell lung cancer risk in the Norwegian subjects after adjustment for age, gender and smoking status. This is the first study to suggest an association between the FAM46A gene VNTR polymorphisms and non small cell lung cancer. We found also that BAG6 rs3117582 SNP was associated with non small cell lung cancer in the Norwegian subjects and the combined Croatian-Norwegian subjects corroborating the earlier finding that BAG6 rs3117582 SNP was associated with lung cancer in Europeans. Logistic regression analyses revealed that genotypes and alleles of BAG6 were independent predictive factor for non small cell lung cancer risk in the Norwegian and combined Croatian-Norwegian subjects, after adjustment for age and gender.